Directed evolution library creation : methods and protocols / edited by Frances H. Arnold and George Georgiou.

Format:

Book

Contributor:

Arnold, Frances Hamilton.

Georgiou, George.

Series:

Methods in molecular biology (Clifton, N.J.) ; v. 231.

Methods in molecular biology ; 231

Language:

English

Subjects (All):

Gene libraries--Laboratory manuals.

Gene libraries.

Genetic engineering--Laboratory manuals.

Genetic engineering.

Molecular evolution--Laboratory manuals.

Molecular evolution.

Microbial mutation--Laboratory manuals.

Microbial mutation.

Combinatorial chemistry--Laboratory manuals.

Combinatorial chemistry.

Genetic recombination--Laboratory manuals.

Genetic recombination.

Gene Library.

Combinatorial Chemistry Techniques.

Medical Subjects:

Gene Library.

Combinatorial Chemistry Techniques.

Genre:

Laboratory manuals.

Physical Description:

x, 224 pages : illustrations (some color) ; 24 cm.

Place of Publication:

Totowa, N.J. : Humana Press, [2003]

Summary:

Directed protein evolution -- a powerful technique for the discovery of new enzymes and therapeutic proteins -- has matured to the point that it is now the centerpiece of enzyme engineering, where it has catalyzed the development of numerous experimental methods. In Directed Evolution Library Creation: Methods and Protocols, seasoned practitioners from many leading laboratories share their best experimental protocols for the generation of molecular diversity. Described in step-by-step detail to ensure experimental success, these protocols include readily reproducible methods for random mutagenesis of entire genes or segments of genes, for homologous and nonhomologous recombination, and for constructing in vivo libraries in bacteria and yeast. In addition to the various protocols for creating libraries, this volume also describes ways to analyze libraries, particularly those made by recombination. An accompanying volume, Directed Enzyme Evolution: Screening and Selection Methods (ISBN: 1-58829-286-X), is devoted entirely to selection and screening methods that can be applied to the directed evolution of enzymes. Taken together, Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods capture for newcomers and more experienced investigators alike all the key methods for using directed protein evolution to better understand protein structure-function relationships, to discover new enzymes and therapeutic proteins, and to design new assays suitable for specific applications.

Contents:

Part I Mutagenesis and Recombination Methods

1 Generating Mutant Libraries Using Error-Prone PCR / Patrick C. Cirino, Kimberly M. Mayer, Daisuke Umeno 3

2 Preparing Libraries in Escherichia coli / Alexander V. Tobias 11

3 Preparing Libraries in Saccharomyces cerevisiae / Thomas Bulter, Miguel Alcalde 17

4 Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP) / Kentaro Miyazaki 23

5 Construction of Designed Protein Libraries Using Gene Assembly Mutagenesis / Paul H. Bessette, Marco A. Mena, Annalee W. Nguyen, Patrick S. Daugherty 29

6 Production of Randomly Mutated Plasmid Libraries Using Mutator Strains / Annalee W. Nguyen, Patrick S. Daugherty 39

7 Evolution of Microorganisms Using Mutator Plasmids / Olga Selifonova, Volker Schellenberger 45

8 Random Insertion and Deletion Mutagenesis / Hiroshi Murakami, Takahiro Hohsaka, Masahiko Sisido 53

9 Random Oligonucleotide Mutagenesis / Jessica L. Sneeden, Lawrence A. Loeb 65

10 Saturation Mutagenesis / Radu Georgescu, Geethani Bandara, Lianhong Sun 75

11 DNA Shuffling / John M. Joern 85

12 Family Shuffling with Single-Stranded DNA / Wenjuan Zha, Tongbo Zhu, Huimin Zhao 91

13 In Vitro DNA Recombination by Random Priming / Olga Esteban, Ryan D. Woodyer, Huimin Zhao 99

14 Staggered Extension Process (StEP) In Vitro Recombination / Anna Marie Aguinaldo, Frances H. Arnold 105

15 RACHITT: Gene Family Shuffling by Random Chimeragenesis on Transient Templates / Wayne M. Coco 111

16 The Creation of ITCHY Hybrid Protein Libraries / Marc Ostermeier, Stefan Lutz 129

17 Preparation of SCRATCHY Hybrid Protein Libraries: Size- and In-Frame Selection of Nucleic Acid Sequences / Stefan Lutz, Marc Ostermeier 143

18 Sequence Homology-Independent Protein Recombination (SHIPREC) / Andrew K. Udit, Jonathan J. Silberg, Volker Sieber 153

19 Producing Chimeric Genes by CLERY: In Vitro and In Vivo Recombination / Valerie Abecassis, Denis Pompon, Gilles Truan 165

Part II Analysis of Library Diversity

20 Analysis of Shuffled Libraries by Oligonucleotide Probe Hybridization / Peter Meinhold, John M. Joern, Jonathan J. Silberg 177

21 Sequence Mapping of Combinatorial Libraries on Macro- or Microarrays: Experimental Design of DNA Arrays / Valerie Abecassis, Gilles Truan, Loic Jaffrelo, Denis Pompon 189

22 Sequence Mapping of Combinatorial Libraries on Macro- and Microarrays: Bioinformatic Treatment of Data / Denis Pompon, Gilles Truan, Valerie Abecassis 199.

Notes:

Includes bibliographical references and index.

ISBN:

1588292851

OCLC:

52037993